Method and reagents for detecting water contamination

ABSTRACT

A method of examining a water supply for microbial contamination involves contacting a water supply with a novel reagent having high specificity for a  Legionella  microbial species, wherein said reagent does not cross-react, or minimally cross-reacts, with a microbial species other than  Legionella . The method further involves detecting, or measuring, a contaminating concentration of a  Legionella  species in the water supply. Useful reagents comprise at least one nucleotide sequence primer comprising a primer sequence selected from SEQ ID NO: 3-10 or a combination of such primer sequences. The increased specificity of such reagents permits more sensitive detection of microbial contamination.

BACKGROUND OF THE INVENTION

Legionella pneumophila is a ubiquitous bacterium associated with freshwater. This microorganism can cause a potentially fatal pneumonia, i.e.,Legionnaire's Disease, pneumonia, Pontiac fever, if inhaled; and thus isa serious problem for owners, operators, and treaters of domestic watersystems, including operators of public facilities which provide water,such as hotels and restaurants, etc. While outbreaks of L. pneumophilainfection associated with cooling water systems are rarer than outbreakslinked to domestic water systems, such as fountains and HVAC-relatedcomponents, the ability of cooling towers to spread water dropletscontaminated with L. pneumophila can cause an outbreak that covers awide geographical area.

To provide guidance to owners, operators, and treaters of coolingsystems various guidelines, codes of practices, and regulations havebeen put into place globally to detect and/or control the spread ofLegionella in water sources. In North America, there are two guidelineswritten by industry groups that are soon to be finalized—the ASHRAE 188Pand the CTI STD-159 standard. In addition, OSHA has recommended actionlevels for remediation and treatment based on counts of Legionella foundin either domestic or industrial water systems.

The ability to identify and/or quantitatively identify Legionella from amixed microbial population in a water source is critical to determinethe potential for infections and to determine if the current treatmentprotocol is effective at either preventing or remediating Legionella inany water system. The US CDC has recommended a culture-based testapproach that leverages the acid-tolerance of Legionella, a distinctivecolony morphology, inherent resistance to the antibiotics glycine,vancomycin, polymixin B and cyclohexamide and the absolute requirementsof addition iron and L-cysteine in growth media to successfully enrichLegionella bacteria from a mixed culture. However, Legionella is also arelatively slow-growing organism that requires 2-10 days under optimalgrowth conditions to appear on growth media. Given the complex mediarequirements to successfully isolate Legionella and the relativelylengthy incubation time needed for each growth step, this method fordetecting Legionella is not optimal.

Other detection technologies take advantage of unique Legionella outermembrane proteins. A latex agglutination method by Oxoid Ltd, UnitedKingdom is a tool for confirming that a Legionella colony is in fact L.pneumophila SG1, the strain most linked to Legionella outbreaks. Othermethods such as Biotica's LEGIPID™ test and Hydrosense's colorimetricantibody test, which has been marketed by Nalco as the Fastpath DuoSystem, are relatively rapid antibody-based detection methods forLegionella in water. For a direct visualization of Legionella in waterthere are fluorescent antibodies that delineate Legionella by serogroup1 and 2-14/15/16 that can be used with a fluorescent microscope. Alimitation of these methodologies and devices is that they requireproteins to detect Legionella. As proteins can exist in water after celldeath, these methods can result in false positive detections.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of qPCR results with qPCR primers.

FIG. 1B is a graph of qPCR showing results of detection of Legionellaspecies other than L. pneumophila vs. L. pneumophila and L. dumoffi.

FIG. 2 is an ompS primer location cartoon.

FIG. 3A is a gel image of primer 327 vs. 528 GC screening againstpurified genomic DNA in Lanes identified as A: DNA ladder, B: L.pneumophila (control), C: Pseudomonas (negative control); D: L.dumoffii; E. L. longbeachae; F: L. cherrii; G: L. pneumophila SG6A; H:L. pneumophila SG6B and I: L. feelei. All species were detected by theprimer showing some cross-reactivity.

FIG. 3B is a gel image of primer 327 SEQ ID NO: 3 and primer 660 GC SEQID NO: 9 screening against purified genomic DNA in Lanes identified asin FIG. 3A. L. feelei was not detected by the primer pair, which showedstronger cross-reactivity.

FIG. 3C is a gel image of primer 327 SEQ ID NO: 3 vs. 825 GC SEQ ID NO:10 screening against purified genomic DNA in Lanes identified as in FIG.3A. L. feelei was not detected by the primer pair, which showed minorcross-reactivity.

FIG. 3D is a gel image of primer 528 SEQ ID NO: 4 vs 825 GC SEQ ID NO:10 screening against purified genomic DNA in Lanes identified as in FIG.3A. All species were detected. Minor cross reactivity was observed.

FIG. 3E is a gel image of primer 327 SEQ ID NO: 3 vs. 660 GC SEQ ID NO:9 screening against purified genomic DNA in Lanes identified as in FIG.3A. All species were detected. Minor Legionella cross reactivity wasobserved. No or minimal Pseudomonas detection was observed.

FIG. 3F is a gel image of primer 660 SEQ ID NO: 5 vs 825 GC SEQ ID NO:10 screening against purified genomic DNA in Lanes identified as in FIG.3A. L. feelei was not detected. Legionella cross reactivity wasobserved. No or minimal Pseudomonas detection was observed.

FIG. 4 is a gel image of the testing of DNA isolated from CDC-ELITEtesting samples with primers 528 SEQ ID NO: 4/825GC SEQ ID NO: 10. TheDNA in Lanes was identified as A: DNA marker, B: CDC-ELITE Sample 1, C:CDC-ELITE Sample 1-conc; D: CDC-ELITE Sample 2, E: CDC-ELITE Sample2-conc; F: CDC-ELITE Sample 3, G: CDC-ELITE Sample 3-conc; H: CDC-ELITESample 4, I: CDC-ELITE Sample 4-conc; J: CDC-ELITE Sample 5, K:CDC-ELITE Sample 5-conc; L: CDC-ELITE Sample 6, M: CDC-ELITE Sample6-conc. Promising results were obtained with unfiltered samples.Background increases with ˜200 fold sample concentration. Conclusionsfrom CDC testing of 528/825GC ompS primers SEQ ID NOs: 4 and 10,respectively, are indicated by the arrows, reading from left to right:

-   -   Arrow 1. Pure L. pneumophila sg 3, 2.6×10³ cfu/ml;    -   Arrow 2. Pure L. pneumophila sg 14, 3.3×10¹ cfu/ml;    -   Arrow 3. Negative for L. pneumophila;    -   Arrow 4. Negative for L. pneumophila;    -   Arrow 5. Mixed L. pneumophila sg 6, 1.3×10³ cfu/ml;    -   Arrow 6. Mixed L. pneumophila sg 6, 6.0×10¹ cfu/ml.

FIG. 5 is a gel image showing comparative testing of DNA isolated fromCDC-ELITE testing samples with inventors' primers (DMC) and knownprimers SEQ ID NO: 15 and 16. The DNA in Lanes was identified as A: DNAmarker, B: CDC-ELITE Sample 1 DMC, C: CDC-ELITE Sample 1-known; D:CDC-ELITE Sample 2 DMC, E: CDC-ELITE Sample 2-known; F: CDC-ELITE Sample3 DMC, G: CDC-ELITE Sample 3-known; H: CDC-ELITE Sample 4 DMC, I:CDC-ELITE Sample 4-known; J: CDC-ELITE Sample 5 DMC, K: CDC-ELITE Sample5-known; L: CDC-ELITE Sample 6 DMC, M: CDC-ELITE Sample 6-known. Theseresults show no meaningful difference with testing samples. Both DMCprimers and known primers (derived from US Patent Publication No.20120171661; SEQ ID NOs: 15 and 16) detect L. pneumophila in mixedmock-environmental samples. No improvement was observed.

FIG. 6A is a gel showing results of comparative testing of DNA isolatedfrom monoculture genomic DNA isolates with DMC primers SEQ ID NO: 4 and10 and SEQ ID NO:15 and 16 primers with P. aeruginosa as a negativecontrol. Both DMC primers and primers SEQ ID NO:15 and 16 detect a widerange of Legionella species. The DMC primers are less cross-reactiveagainst high levels of contaminating DNA relative to P. aeruginosa. Thelanes of the gel are identified on the gel.

FIG. 6B is a gel showing comparative testing of DNA isolated frommonoculture genomic DNA isolates with DMC primers SEQ ID NOs: 4 and 10,and the known primers SEQ ID NOs: 15 and 16 with P. dentrificans as anegative control. 2 μl of PCR product was loaded onto a 2.2% LonzaFLASHGEL™ system. The lanes A-K of the gel are as follows: A: DNAladder, B: L. pneumophila (DMC), C: L. pneumophila (known); D: P.denitrificans (DMC); E: P. denitrificans (known); F: L. dumoffii (DMC);G: L. dumoffii (known); H: L. longbeachae (DMC); I: L. longbeachae(known); J: L. feelei (DMC); and K: L. feelei (known). The SEQ ID NO: 4and 10 PCR primers demonstrate an improvement over known primers. Thesequences described herein provide less-cross reactive detection ofLegionella species under a variety of conditions.

FIG. 7 is a depiction of the L. pneumophila ompS gene sequence SEQ IDNO: 17 indicating the nucleotide locations of the novel primers SEQ IDNOs: 1-10 and other known primers SEQ ID NOs: 15 and 16 discussedherein.

FIG. 8A is a gel image of Omp528/825GC SEQ ID NOs: 4/10 primers showinglow-concentration gradient testing against P. aeruginosa ATCC 15442. Thelanes A-M of the gel are as follows:

-   -   A: DNA ladder,    -   B: 1×10⁵ cfu/ml non-target DNA,    -   C: 1×10⁵ cfu/ml non-target DNA, 1×10¹ cfu/ml L. pneumophila        BAA74 DNA;    -   D: 1×10⁵ cfu/ml non-target DNA, 1×10² cfu/ml L. pneumophila        BAA74 DNA;    -   E: 1×10⁵ cfu/ml non-target DNA, 1×10³ cfu/ml L. pneumophila        BAA74 DNA;    -   F: 1×10⁵ cfu/ml non-target DNA, 1×10⁴ cfu/ml L. pneumophila        BAA74 DNA;    -   G: 1×10⁵ cfu/ml non-target DNA, 1×10⁵ cfu/ml L. pneumophila        BAA74 DNA;    -   H: 1×10⁴ cfu/ml non-target DNA, 1×10⁶ cfu/ml L. pneumophila        BAA74 DNA;    -   I: 1×10³ cfu/ml non-target DNA, 1×10⁷ cfu/ml L. pneumophila        BAA74 DNA;    -   J: 1×10² cfu/ml non-target DNA, 1×10⁷ cfu/ml L. pneumophila        BAA74 DNA;    -   K: 1×10¹ cfu/ml non-target DNA, 1×10⁵ cfu/ml L. pneumophila        BAA74 DNA;    -   L: 1×10⁵ cfu/ml L. pneumophila BAA74 DNA;    -   M: No DNA Control.

FIG. 8B is a gel image of Omp528/825GC SEQ ID NOs: 4/10 primers showinglow-concentration gradient testing against Burkholderia cepacia ATCC25416. The lanes A-M of the gel are as in FIG. 8A.

FIG. 8C is a low-concentration gradient testing of Omp528/825GC SEQ IDNOs: 4/10 primers against Sphingomonas paucimobilis BAA 1092. The lanesA-M of the gel are as in FIG. 8A.

FIG. 8D is a low-concentration gradient testing of Omp528/825GC SEQ IDNOs: 4/10 primers against Enterobacteria aerogenes ATCC 13048. The lanesA-M of the gel are as in FIG. 8A.

FIG. 8E is a first low-concentration gradient testing of Omp528/825GCSEQ ID NOs: 4/10 primers against Klebsiella pneumonia ATCC 8308. Thelanes A-M of the gel are as in FIG. 8A.

FIG. 8F is a repeated low-concentration gradient testing of Omp528/825GCSEQ ID NOs: 4/10 primers against Klebsiella pneumonia ATCC 8308. Thelanes A-M of the gel are as in FIG. 8A.

FIG. 9A is a low-concentration gradient testing of known 1116R/492 SEQID NOs: 12/11 primers against P. aeruginosa ATCC 15442. The lanes A-M ofthe gel are as in FIG. 8A. PCR conditions with 1 μM primer, 3 mins 95 C;35× (94 C at 30 s, 50 C at 30 S, 72 C 30 s and 10 min 72 C.

FIG. 9B is a low-concentration gradient testing of 1116R/492 SEQ ID NOs:12/11 primers against Burkholderia cepacia ATCC 25416. The lanes A-M ofthe gel are as in FIG. 8A. PCR conditions are as in FIG. 9A.

FIG. 9C is a low-concentration gradient testing of 1116R/492 SEQ ID NOs:12/11 primers against Sphingomonas paucimobilis BAA 1092. The lanes A-Mof the gel are as in FIG. 8A.

FIG. 9D is a low-concentration gradient testing of 1116R/492 SEQ ID NOs:12/11 primers against Enterobacteria aerogenes ATCC 13048. The lanes A-Mof the gel are as in FIG. 8A.

FIG. 9E is a repeated low-concentration gradient testing of 1116R/492SEQ ID NOs: 12/11 primers against Klebsiella pneumonia ATCC 8308. Thelanes A-M of the gel are as in FIG. 8A.

FIG. 10A is a low-concentration gradient testing of 1126R/450 SEQ IDNOs: 14/13 primers against P. aeruginosa ATCC 15442. The lanes A-M ofthe gel are as in FIG. 8A.

FIG. 10B is a gel image of 1126R/450 SEQ ID NOs: 14/13 primers showinglow-concentration gradient testing against Burkholderia cepacia ATCC25416. The lanes A-M of the gel are as in FIG. 8A.

FIG. 10C is a low-concentration gradient testing of 1126R/450 SEQ IDNOs: 14/13 primers against Sphingomonas paucimobilis BAA 1092. The lanesA-M of the gel are as in FIG. 8A. See Table 2 for the cycle conditions.

FIG. 10D is a low-concentration gradient testing of 1126R/450 SEQ IDNOs: 14/13 primers against E. aerogenes ATCC 13048. The lanes A-M of thegel are as in FIG. 8A.

FIG. 11A is a low-concentration gradient testing of US20120171661primers SEQ ID NO: 15 and 16 against P. aeruginosa ATCC 15442. The lanesA-M of the gel are as in FIG. 8A. PCR conditions are listed in Table 2.

FIG. 11B is a gel image of F and R known primers SEQ ID NOs: 15 and 16showing low-concentration gradient testing against Burkholderia cepaciaATCC 25416. The lanes A-M of the gel are as in FIG. 8A.

FIG. 11C is a low-concentration gradient testing of US20120171661 F andR primers SEQ ID NOs: 15 and 16 against Sphingomonas paucimobilis BAA1092. The lanes A-M of the gel are as in FIG. 8A. See Table 2 for thecycle conditions.

FIG. 11D is a low-concentration gradient testing of US20120171661 F andR primers SEQ ID NOs: 15 and 16 against E. aerogenes ATCC 13048. Thelanes A-M of the gel are as in FIG. 8A.

FIG. 11E is a low-concentration gradient testing of US20120171661 F andR primers SEQ ID NOs: 15 and 16 against Klebsiella pneumonia ATCC 8308.The lanes A-M of the gel are as in FIG. 8A.

SUMMARY OF THE INVENTION

In one aspect, a method of examining a water supply for microbialcontamination is described which employs novel sensitive reagents havinghigh specificity for a Legionella microbial species. This method employsreagents that do not cross-react, or minimally cross-reacts, with amicrobial species other than Legionella. The method involves contactinga water supply with the reagent and detecting, or measuring, acontaminating concentration of a Legionella species in the water supply.In one embodiment, the method employs PCR or qPCR steps includingannealing a reagent primer or a combination of primers to a targetednucleic acid sequence present in Legionella at a selected annealingtemperature. In another embodiment, the method employs hybridizationbased steps. The method permits the determination of whether Legionellaconcentration is within acceptable safety limits.

In another aspect, a novel reagent having high specificity for aLegionella microbial species, e.g., L. pneumophila, and which does notcross-react, or minimally cross-reacts, with a microbial species otherthan Legionella is provided. In one embodiment, the reagent comprisesnucleotide primers, such as qPCR or PCR primers, with high specificityfor multiple Legionella species, but not for Pseudomonas species. Inanother embodiment, the reagent includes a substrate upon which one ormore of the nucleotide sequences are immobilized or fixed. In anotherembodiment, the reagent is a biosensor containing primers identifiedherein.

In yet another embodiment, a kit comprising one or more of the novelreagents described herein and other assay method components includinglabels, substrates, a label component capable of interacting with thelabel and generating a detectable signal, is provided.

Other aspects and advantages of these methods and compositions aredescribed further in the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

As described herein, novel compositions, e.g., reagents, and methods areprovided that enable a fast and accurate detection of microbialcontamination of Legionella, e.g., Legionella pneumophila, in watersamples or supplies. As demonstrated in the examples below, thecompositions (reagents and primers) and methods described herein providean improvement in detection methods and compositions which have beenused for Legionella detection. The reagents and primers described hereinare highly specific to Legionella, and employ target sequences largelyoverlooked relative to more common detection genes such as mip ordot/icm. These primers are employed in methods of detection ofcontamination that employ qPCR-based detection of Legionella species,preferably L. pneumophila species, PCR and hybridization-basedmethodologies. The methods and the primers used therein allow lowertemperatures to be employed in primer annealing, which providesefficiencies in energy use during performance of the methods. Furtherthe reagents and methods described herein have less cross-reactivityagainst high levels of contaminating DNA, e.g., Pseudomonas, which iscommon in water systems, and these methods and compositions therebyaccomplish the detection of Legionella contamination with less falsepositive results.

A. Definitions and Components of the Methods and Compositions

By the term “water supply” or “samples” as used herein is meant anynaturally occurring bodies of water, e.g., lakes, streams, rivers, orindustrial or domestic artificial container or body carrying water,e.g., reservoirs, pools, fountains, potable or drinking water, HVACsystems containing or carrying water, bottled water, industrial watersupplies or containers, waste water containers, etc. These watersupplies or samples can contain purely Legionella, a mixed population ofmicrobial organisms with high Legionella levels, a mixed population ofmicrobial organisms with low Legionella levels, a mixed population ofmicrobial organisms with no Legionella, or no microbial contamination.Such samples or water supplies can contain unconcentrated orconcentrated DNA samples of a target sequence from the indicatedmicroorganism.

By “target” is meant a nucleic acid sequence that is found in one ormore Legionella species and has certain desirable characteristics. Suchcharacteristics include relatively stable expression at relatively highlevels compared to other Legionella gene sequences. Anothercharacteristic is that the nucleotide sequence expression is insensitiveto growth conditions and sufficiently unique to Legionella to preventdetection of non-Legionella microorganisms. The target sequence may befound in nucleic acid materials from the Legionella microorganisms thatinfect mammalian subjects, e.g., humans, and contaminate water or watersupplies. In one embodiment, the target is a sequence encodes portionsof the Legionella major outer membrane protein. The target is asingle-stranded ribonucleic acid sequence, or a single strand of adeoxyribonucleic acid sequence from Legionella. Included in thisdefinition are RNA, mRNA, microRNA, a single strand of DNA, cDNA,small-interfering RNA (siRNA), short-hairpin RNA (shRNA), peptidenucleic acid (PNA), transfer RNA (tRNA), ribosomal RNA (rRNA) and DNAfrom a Legionella gene. In one embodiment, the selected target gene isknown as the major outer membrane protein gene of Legionella, or ompS,omp28, lpg1974 or lpnomp28. In one embodiment, the target, ompS, is agene that appears to be stably expressed regardless of growth conditionand is heavily conserved within the Legionella genus, making it anappealing target for detection of water supply contamination. Thenucleotide sequence of the ompS gene can be found in the Genbankdatdabase at accession No. M76178. See also, SEQ ID NO: 17. This targetgene also includes the mompS or major outer membrane protein precursorgene, the sequences of which is publically available in the Genbankdatabase at accession No. AF078147.

More specifically, the target of the ompS sequences may be nucleic acidregions of SEQ ID NO: 17, or a homolog or naturally occurring orthologof same that provides a suitable “target” for detection by anucleic-acid based detection tool, such as qPCR primers, PCR primers orhybridization probes. For ease of use in identifying target regions ofompS, this specification will refer to regions of SEQ ID NO: 17.However, it is understood by one of skill in the art that homologoussequences of Legionella ompS genes having modifications or mutationsfrom the sequence of SEQ ID NO: 17 or from other Legionella species ompSare also included in the descriptions of the reagents and methodsherein.

By “Legionella” is meant any species of the genus Legionella,particularly those species commonly found in bodies of water or watersupplies. Legionella species include without limitation, L. pneumophila,L. dumoffii, L. longbeachae, L. cherrii, L. pneumophila SG6A, L.pneumophila SG6B, L. feelei or combinations thereof. It is a goal of thereagents and methods described herein to detect contaminating L.pneumophila, among other species, because it causes human disease.

The term “reagent” as used herein can refer to a single primer, one ormultiple sets of forward and reverse primers, labeled primers, primersimmobilized on a substrate or in an array or microarray, or devicesincorporating any of the above for use in detecting Legionallacontamination in various water supplies.

The term “primer” as used herein is intended to mean oligonucleotidesequences that can bind to and amplify the target sequence in theperformance of PCR or qPCR. In one embodiment, a primer set comprises aforward primer that binds to the coding strand of the target in the 5′to 3′ direction. In another embodiment, a primer set comprises a reverseprimer that binds to the complement of the target sequence in the 3′ to5′ direction. As used herein the primers may be about 15 to about 50nucleotides in length, including any intermediate length in-between,including at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49 or 50 nucleotides in length. Such primers may be employed insolution, e.g., in buffer, or immobilized on a substrate or microarray.In other embodiment, the primers may be labeled with detectable, e.g.,fluorescent, labels, e.g., according to the TaqMan® methodology so thatthe target-specific oligonucleotides produce a fluorescent signal onlywhen the target DNA is amplified during qPCR, or using fluorescent orother labels such as SYBR® Green I dye.

The term “microarray” refers to an ordered arrangement of hybridizablearray elements. In one embodiment, a microarray comprises polynucleotideprobes that hybridize to the specified target sequence, on a substrate.In another embodiment, a microarray comprises multiple primers (reverseand forward), optionally immobilized on a substrate.

The term “PCR array” refers to microfluidics card or multiwell platecontaining an ordered arrangement of gene-specific forward and reverseprimers and fluorescence-labeled probe in each micro-well. This array isused with the sample from the water supply, or a product derivedtherefrom (e.g., cDNA or cRNA derived from RNA) in real-time PCRreactions which are monitored using fluorescent dye, for example SYBRGreen or Taqman technology using a reporter fluorescent dye. Each set ofprimers is designed to amplify the target sequence of interest, such asthose described herein. Such primers can readily be designed by one ofskill in the art using known techniques, given the instructions on theprimer identities described in this specification. See e.g., reference13. Primers may also be prepared or available commercially, for examplefrom Invitrogen:http://bioinfo.invitrogen.com/genome-database/browse/gene-expression/keyword/Taqman%20primers?ICID=uc-gex-Taqman.

The term “real-time quantitative PCR”, or “qPCR” refers to a techniquecombining PCR amplification and detection into a single step. With qPCR,fluorescent dyes are used to label PCR products during thermal cycling.Real-time PCR instruments measure the accumulation of fluorescent signalduring the exponential phase of the reaction for fast, precisequantification of PCR products and objective data analysis. qPCRreaction products are fluorescently labeled using one of two mainstrategies: Reactions are run in real-time PCR instruments with thermalcycling and fluorescence detection capabilities. By using highlyefficient PCR primers and optimal conditions for amplification, everytarget molecule is copied once at each cycle and data are capturedthroughout the thermal cycling. Since qPCR reactions are set up with alarge molar excess of PCR primers and thermostable DNA polymerase, inthe early rounds of thermal cycling, target-template is the limitingfactor for the reaction thus, fluorescent signal is directlyproportional to the amount of target in the input sample.

B. Reagents of the Invention

In one embodiment, a reagent for examining a water supply for microbialcontamination has high specificity for a Legionella microbial species,and does not cross-react, or minimally cross-reacts, with a microbialspecies other than Legionella. In one embodiment, such a reagentcomprises at least one nucleotide sequence primer (DNA or RNA) thatamplifies or hybridizes to a target sequence in Legionella. In anotherembodiment the target sequence is found between and includingnucleotides 528 and 844 of SEQ ID NO: 17. In one embodiment, the ompStarget sequence is found between and including nucleotides 327 and 491of SEQ ID NO: 17. In another embodiment the target sequence is foundbetween and including nucleotides 327 and 844 of SEQ ID NO: 17. Inanother embodiment the target sequence is found between and includingnucleotides 327 and 491 of SEQ ID NO: 17. In another embodiment thetarget sequence is found between and including nucleotides 327 and 550of SEQ ID NO: 17. In another embodiment the target sequence is foundbetween and including nucleotides 327 and 680 of SEQ ID NO: 17. Inanother embodiment the target sequence is found between and includingnucleotides 528 and 550 of SEQ ID NO: 17. In another embodiment thetarget sequence is found between and including nucleotides 528 and 680of SEQ ID NO: 17. In another embodiment the target sequence is foundbetween and including nucleotides 660 and 844 of SEQ ID NO: 17. Inanother embodiment the target sequence is found between and includingnucleotides 660 and 680 of SEQ ID NO: 17. In still other embodiments,the target regions may include those regions identified above includingfrom about 5 to about 15 additional nucleotides on either end of theidentified sequences.

These reagents may be primers as defined above which are selected fromthe forward primer sequences set out in Table 1 below as SEQ ID NOs: 1,3, 4, 5 and 6. It should be understood that these sequences may differfrom those of Table 1 by certain modifications. In one embodiment, amodification is indicated by the presence of the indicated wobble basesshown and defined in Table 1. Other modifications of the primersequences of Table include primer sequences that differ from those ofTable 1 by including from 1 to about 5 additional contiguous nucleotides5′ to the 5′ nucleotide base of each forward primer location in SEQ IDNO: 17. In another embodiment additional modified primer sequences maydiffer from those of Table 1 by including from 1 to about 5 additionalcontiguous nucleotides 3′ to the 3′ nucleotide base of each forwardprimer located in SEQ ID NO: 17. By resort to FIG. 7, one can see howthe identified primers may be modified by adding contiguous bases toeither end of the identified primer, or deleting one or more bases fromeither end of the primer, or a combination of both types ofmodifications. In yet a further embodiment, a primer may differ from asequence of Table 1, by deleting 1, 2, 3, 4, or 5 bases from the 5′ or3′ end of the related sequence in Table 1 and adding 1, 2, 3, 4, or 5additional contiguous bases to the opposite end of the primer, thusshifting the primer sequence. For example, one primer sequence could bea modification of SEQ ID NO:4 and span nucleotides 526 to 550 of SEQ IDNO: 17 or 526 to 547 of SEQ ID NO: 17 and so on.

These reagents may be primers as defined above which are selected fromthe reverse primer sequences set out in Table 1 below as SEQ ID NOs: 2,7, 8, 9, 10. It should be understood that these sequences may differfrom those of Table 1 by modifications such as the indicated wobblebases in that table. As described above, additional modifications canresult in modified reverse primer sequences that differ from those ofTable 1 by including from 1 to about 5 additional contiguous nucleotides5′ to the 5′ nucleotide base of each reverse primer in SEQ ID NO: 17. Inanother embodiment additional reverse primer sequences may differ fromthose of Table 1 by including from 1 to about 5 additional contiguousnucleotides 3′ to the 3′ nucleotide base of each reverse primer in SEQID NO: 17. In a manner analogous to that described above for the forwardprimers, the reverse primers of Table 1 may be modified by shifting theprimer sequence 1 to 5 bases 5′ or 3′ from the primers position on SEQID NO: 17.

Thus, in one embodiment, the reagent comprises a set of primerscomprising a forward nucleotide sequence primer and a reverse nucleotidesequence primer selected from Table 1. In one embodiment, the reagentcontains a forward primer comprising at least one of SEQ ID NO: 1, 3, 4,5 or 6 and the reverse primer comprises at least one of SEQ ID NO: 2, 7,8, 9, or 10. In another embodiment, the reagent contains the forwardprimer comprising SEQ ID NO: 4 and the reverse primer comprising one ofSEQ ID NO: 8, 9 or 10. In another embodiment, the reagent contains theforward primer comprising SEQ ID NO: 4 and the reverse primer comprisingSEQ ID NO: 10. In another embodiment, the reagent contains the forwardprimer comprising SEQ ID NO: 4 and the reverse primer comprising one ofSEQ ID NO: 8. In another embodiment, the reagent contains the forwardprimer comprising SEQ ID NO: 4 and the reverse primer comprising one ofSEQ ID NO: 9. In a similar manner, the reagent may comprise othercombinations of the forward and reverse primers of Table 1, SEQ ID NOs:1-10. In still another embodiment, the reagent comprises multiple setsof reagents selected from the primers comprising SEQ ID NOs: 1-10 or3-10. In still another embodiment, the reagent comprises all nucleotidesequence primers selected from primer sequences comprising SEQ ID NO:1-10, or sequences having modifications thereof on the 5′ or 3′ ends asdescribed above.

In still another embodiment, a reagent comprises a substrate upon whichone or more of the nucleotide sequences are immobilized or fixed. Such asubstrate is an array, a microarray, a microchip, a plastic surface, ora glass surface. Association of the primer on the substrate and methodsfor accomplishing the association are well known in the art.Alternatively, the primer sequences may be provided in a suitable bufferdepending upon the type of method in which the reagent is to be used.

In still another embodiment, the reagent includes a primer to which adetectable label or label component is associated. Suitable detectablelabels include fluorescent labels such as those employed by the TAQMANreagents, or other known fluorescent molecules. Suitable labels may beselected from among many known label types by one of skill in the artgiven the teachings of this specification.

In yet another embodiment, a reagent of this invention can be in theform of a kit containing one or more of the primers, reverse and forwardprimer sets or labeled primer-probes, suitable labels and labelingmethodologies, suitable substrates, and/or label component, e.g.,enzymes, capable of interacting with the label associated with a primerand generating a detectable signal.

In yet another embodiment, the reagent is configured as a watermonitoring device comprising the primers described herein.

C. Methods of Detecting Contamination

The reagents, e.g., primers described herein, are desirably used inmethods for Legionella detection involving the techniques of polymerasechain reaction (PCR), quantitative PCR (qPCR), or oligonucleotidehybridization-based methodologies.

In one aspect, a method of examining a water supply for microbialcontamination, particularly contamination with Legionella, is provided.Such a method involves contacting a water supply with a reagent havinghigh specificity for a Legionella microbial species, wherein the reagentdoes not cross-react, or minimally cross-reacts, with a microbialspecies other than Legionella. Thereafter, the method involvesdetecting, or measuring, a contaminating concentration of a Legionellaspecies in the water supply.

In one embodiment, the reagent employed in the method is one of thereagents described herein. For example, the method can employ a reagentcomprising at least one nucleotide sequence primer selected from:

-   -   (a) forward primer sequence 327 comprising SEQ ID NO: 3    -   (b) forward primer sequence 528 comprising SEQ ID NO: 4    -   (c) forward primer sequence 660 comprising SEQ ID NO: 5;    -   (d) forward primer sequence 825 comprising SEQ ID NO: 6    -   (e) reverse primer sequence GC-327 comprising SEQ ID NO: 7;    -   (f) reverse primer sequence GC-528 comprising SEQ ID NO: 8    -   (g) reverse primer sequence GC-660 comprising SEQ ID NO: 9;    -   (h) reverse primer sequence GC-825 comprising SEQ ID NO: 10; or    -   (i) a combination of primer sequences comprising (a) through (h)        or    -   (j) modifications of such primer sequences with wobble bases or        by adding or deleting one or more 5′ or 3′ bases or by shifting        the position of the primer 1 to 5 bases along its position in        SEQ ID NO: 17.

In another embodiment, the method employs multiple sets of reagentsselected from the primers of (a) through (j), or includes multiple stepsusing some or all of the primers or reverse/forward primer pairs in asuitable methodology. Any of the reagents described above are believedto be useful in the methods described below and in the examples.

Thus, in one embodiment, the method involves performing PCR or qPCRsteps to amplify the target sequence by annealing a primer or acombination of primers to the targeted nucleic acid sequence present inLegionella at a selected annealing temperature. In one embodiment, theannealing temperature is less than 58° C. In another embodiment, theannealing temperature is about 40° C. In another embodiment, theannealing temperature is about 41° C. In another embodiment, theannealing temperature is at least 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50° C. In still other embodiments, the annealing temperature ofthe reagents described herein is lower than the annealing temperaturesemployed by use of other known ompS primers. For example, as shown inTable 2, the method can involve contacting a sample with 1 μM primer for3 min at 95° C.; and performing 35 cycles of denaturation for 30 sec at94° C.; annealing the primers for 30 sec at 40° C.; elongation for 30sec at 72° C.; and a 10 min 72° C. extension step. Still otherconditions are described in detail in the examples below.

In another embodiment, the measuring comprises hybridizing a primer to atargeted nucleic acid sequence present in Legionella and performing apolymerase chain reaction (PCR) or a quantitative polymerase chainreaction (qPCR). These methods employ amplifying the targeted sequenceand determining whether said Legionella concentration is withinacceptable safety limits.

A variety of methods of measuring or detecting the amount ofcontamination are known in the art once the primers have amplified thetarget sequence or identified in by hybridization. Methods of detectingcan include running the amplified or hybridized target sequences on withagarose gel for visualization. Running electric current through the gelcauses the negatively-charged DNA to migrate to positively charged sideof gel. The gel is stained with dye that binds to double-stranded DNA,thereby allowing visualization. The larger the DNA, the slower it movesthrough the gel. Still other methods of measuring the results of PCR andhybridization methodologies can be selected by one of skill in the art.

Thus, in one embodiment, the methods employ a reagent with highspecificity for multiple Legionella pneumophila only. Thus, in oneembodiment, the method and reagents can distinguish betweencontamination of a water supply with Legionella from contamination witha Pseudomonas species, such as Pseudomonas aeruginosa or Pseudomonasdenitrificans.

As demonstrated in the examples below, the primers described hereinprovide a higher degree of target specificity in qPCR and PCRmethodologies. The examples showed testing against purified DNA fromvarious strains, and testing against DNA extracted from six CDC ELITEtesting samples. As discussed below, primers 528/825gc are better thanthe known SEQ ID NO: 15 and 16 primer sequences described in US patentpublication No. 20120171661 because they have decreased cross-reactivitywith other species of microorganism. These qPCR primers, e.g., SEQ IDNO: 1 and 2 or 4 and 10 allow for faster detection of Legionella thanthe primers disclosed in other known assays, such as that of US patentpublication No. 2012071661.

In order to further understand the above-described device and todemonstrate how the method may be carried out in practice, certainembodiments will now be described with reference to the accompanyingdrawings above and the examples described below. The following examplesare provided for illustration and do not limit the disclosure or scopeof the claims and specification.

Example 1: Identification of Target DNA and Assays Used

The inventors a Legionella gene identified as lpg1974 (Weissenmayer,Prendergast et al. 2011) as a source of a suitable target for novelLegionella detection reagents. This gene, also known as ompS (NCBIGenbank Accession No. M76178.1) was found to be fairly stably expressedat relatively high levels to other Legionella genes. Thus, this gene andits encoding outer membrane protein met the inventors' twin goals of anucleotide sequence whose expression would be insensitive to growthconditions and unique to Legionella to prevent detection of non-targetorganisms.

A. Developing and Testing qPCR Primers

Because the 5′end of ompS appeared to be less variable relative to otherregions of the gene and appeared to be more specific to L. pneumophilaorganisms, it was targeted for qPCR primer generation. The inventorsemployed a tool, i.e., the IDT primer design website(http://www.idtdna.com/scitools/Applications/RealTimePCR/) to constructqPCR primers with specific melting temperature, length, andhomodimer/heterodimer features (Table 1).

Amplification of target DNA was done under the following conditions:

95° C. for 3 minutes; followed by 41 cycles of: 95° C. for 10 seconds,58° C. for 10 seconds, 72° C. for 30 seconds; then 95° C. for 10 s,followed by a melt curve using software default conditions. qPCRamplification was done in the CFX96 Bio-Rad System with a commerciallypurchased Legionella genomic standard used for run validation. The qPCRconditions varied from the recommended instructions, but as efficiencyand R̂2 values were within normal parameters, it is unlikely that thechanges had any effect. These conditions involved using per reactionwell 12.5 μl Bio-Rad iQ SYBR Master Mix, 0.15 μl of both forward andreverse primer (stock primer concentration was 50 μM), and 4.7 μlmolecular-biology grade H₂O (instead of the correct 7.2 μl H₂O). To thatvolume 5 μl of 1/100 diluted purified genomic DNA was added.

B. Developing PCR Primers

Regions of DNA that could be used either for traditional PCR approachesor a hybridization-based approach were also analyzed for a broaderdetection, e.g., of multiple Legionella species. To do this, the DNAsequence of the ompS gene from L. pneumophila and the ompS gene from L.longbeachae were aligned and directly compared to identify regions oftotal similarity or sufficiently high sequence similarity to permit aprimer with 1-2 wobble bases to be identified. “Wobble bases” refer tocertain nucleotide bases located in a primer sequence for whichalternative base options are possible, based upon the primer pool usedin a reaction. For example, a wobble base of “R” would be 50% guanineand 50% cytosine in a given primer pool. This approach allowed for thebroadening of a primer reactivity to include highly related but not 100%identical regions of DNA.

The ompS alignment was visually examined to identify 4 regions of DNAthat could be used for primers, with complementary primers generated sothat key regions could be used for different tests. The primersidentified in Table 1 below, were tested using a traditional PCR 2×Master Mix (Promega) with the following conditions: 12.5 μl Master Mix,0.5 μl of both forward and reverse primers, and 9.5 μl dH₂O. To this, 2μl of purified genomic DNA isolated using the MolBio Ultraclean DNAIsolation Kit was added. The PCR conditions in the initial screen wereas follows: 2 min at 95° C., 35 cycles of: 30 s at 95° C., 30 s at 45°C. (40° C. for primers 528 SEQ ID NO: 4 and 825Gc SEQ ID NO: 10 due toprimer melting temperature requirements), and 30 s at 72° C.; then 10min at 72° C. for a final extension step.

The primer sequences SEQ ID NO: 1-10 and known sequences SEQ ID NO: 15and 16 identified in Table 1 are located on the ompS gene as illustratedin FIG. 7.

TABLE 1 PRIMER SEQUENCES AND NAMES Inventor's  SEQ Primer ID SequencesSequence⁴ Target Appln # Legionella OmpS GCA GTG CTT TGT ompS qPCR  1Set 3 Forward TTG CAG GTA CGA Legionella OmpS ATG CCA ATT TCT ompS qPCR 2 Set 3 Reverse CCA GCC ACC AAC OmpS 327F TTT GCA GGT ACS ompS PCR  3ATG GGT CCA GT OmpS 528F GAA GGT TCT TAT ompS PCR  4 CAC TTC AAC ACOmpS 660F GAA MTG GGK CAA ompS PCR  5 TTY GTT GAT OmpS 825FATG AAY TAT GTA ompS PCR  6 TTY GGT AA GC-OmpS 327F⁵ ACT GGA CCC ATSompS PCR  7 GTA CCT GCA AA GC-OmpS 528F⁵ GTG TTG AAG TGA ompS PCR  8TAA GAA CCT TC GC-OmpS 660F⁵ ATC AAC RAA TTG ompS PCR  9 MCC CAK TTCGC-OmpS 825F⁵ TTA CCR AAT ACA ompS PCR 10 TAR TTC ATPrimer Sequences obtained from other publications mompS-492F¹GACATCAATGTGAAC mompS PCR 11 TGG mompS-1116R¹ TGGATAAATTATCCA mompS PCR12 GCCGGACTTC mompS-450F² TTG ACC ATG AGT mompS PCR 13 GGG ATT GGmompS-1126R² TGG ATA AAT TAT mompS PCR 14 CCA GCC GGA CTT CU520120171661F³ gcg gct gta ttt ompS PCR 15 gct ctg gga aU520120171661R³ taa gcc tat gta ompS PCR 16 ggg gcc aga tgc ¹Source isreference no. 11. ²Source is reference no. 10 ³Source is reference no.8. ⁴Nucleic acid “wobble base” abbreviations include S representingeither C or G; M representing either A or C; Y representing either C orT; R representing either A or G; and K representing either G or T. ⁵The“gc” on the primer name refers to “generated complement”, where theprimer manufacturer (IDT) generated a primer that was located on thesame location on the opposite strand of DNA so that the same region ofDNA could be tested with primers both 5′ and 3′ to its location.

C. Initial Primer Comparison

A series of benchmarking of known primers was conducted to identifywhether the novel ompS primers demonstrated an improvement when comparedwith other known ompS primers. In one comparison at conditions that werebest suited for the primer setup (2° C. below the primer meltingtemperature (TM), the reaction conditions were 12.5 μl Master Mix, 0.5μl of both forward and reverse primers, and 10.5 μl dH₂O where 1 μl ofpurified genomic DNA isolated using the MolBio Ultraclean DNA IsolationKit was added. DNA from defined strains and from nucleic acid isolatedfrom a CDC ELITE testing panel were assayed.

D. Refined Primer Comparison

Cell suspensions from defined bacterial species (Legionella pneumophilaATCC BAA74, P. Pseudomonas aeruginosa ATCC 15442, Burkolderia cepaciaATCC 25416, Sphingomonas paucimobilis BAA1092, Flavobacterium odoratum(aka Myroides odoratus) NCIMB 13294, Enterobacter aerogenes ATCC 13048,and Klebsiella pneumonia ATCC 8308) were obtained and split into twoaliquots. One aliquot was plated to obtain a cfu/ml count as a measureof organism concentration and the other was subjected to DNA isolationvia the MolBio UltraClean DNA Kit. Once the cfu/ml of each startingcolony was identified, each DNA vial was assigned an equivalent genomicunit/ml concentration based on the cell counts. From that, a dilutionrange was generated to simulate absence of target organism, thenincreasing amounts of Legionella relative to a high level of non-targetorganism DNA (see conditions specified in Table 2).

TABLE 2 PRIMER EXPERIMENTAL CONDITIONS Primer Rxn PCR Cycle Conditions(DNA in water; mimics Test Sequences Target Mix low concentrationsamples) qPCR SEQ ID ompS qPCR 0.333 μM primer (ideally 0.3 μM); 95° C.for 3 min, test NOs: 1 41 cycles of denaturation for 10 sec at 95° C.;and 2 annealing for 10 sec at 58° C.; elongation for 30 sec at 72° C.;10 sec at 95° C.; a melt curve analysis SYBR green detection Primer SEQID ompS PCR 1 μM primer, 3 min at 95° C.; 35 cycles of Conditions NOs: 4denaturation for 30 sec at 94° C.; annealing for 30 and 10 sec at 40°C.; elongation for 30 sec at 72° C.; 10 min 72° C. extension stepComparison SEQ ID mompS PCR 1 μM primer, 35 cycles of denaturation for30 sec Test 1 NOs: 11 at 94° C.; annealing for 30 sec at 50° C.;elongation and 12 for 30 sec at 72° C.; 10 min 72° C. extension stepComparison SEQ ID mompS PCR 0.2 μM primer; 35 cycles of denaturation for30 Test 2 NOs: 13 sec at 95° C.; annealing for 30 sec at 55° C.; and 14elongation for 40 sec at 72° C.; 10 min 72° C. extension step ComparisonSEQ ID ompS PCR 1 μM primer; 35 cycles of denaturation for 60 sec Test 3NOs: 15 at 94° C.; annealing for 60 sec at 58° C.; elongation and 16 for90 sec at 72° C.; 10 min 72° C. extension step

A master block was made with the DNA concentrations so that the same DNAmaterial would be used in each reaction. To confirm that no meaningfulchanges in the DNA sample or that false-positives occurred fromcross-contamination from the start of the reaction to the end, theprimers SEQ ID NOs: 1-10 were tested at the beginning and towards theend of the experiment. Primers were tested at the conditions suggestedin other publications.

E. DNA Gel Protocol

To visualize PCR fragments either Lonza's Flashgel cassette-based systemor a standard operating procedure was followed for gel casting, buffer,and safety considerations. According to the SOP, 5 μl of PCRproduct/loading dye was added to each well with 5 of 100 bp DNA Ladder(Promega)/loading dye added to the left most well (2 μl for the LonzaFlashgel system). 1.5% agarose gels were run in 1×TBE buffer for 30minutes at 100V. Then, they were placed into an empty plastic pipettetip box and exposed to 50 ml of 1×SYBR Green (Invitrogen) forapproximately 60 minutes, then visualized on the Bio-Rad Gel XR Imager.Lonza Flashgels were operated using manufacturer's instructions andvisualized with the built-in imaging camera.

Example 2: qPCR-Based Method for Broad Legionella Detection

qPCR primers SEQ ID NOs. 1 and 2 were generated to determine whether theompS gene could be used in a qPCR-based method for broad Legionelladetection. DNA from liquid culture-grown P. aeruginosa and P.denitrificans was used as negative controls. DNA from L. pneumophilaBAA74 served as a positive control, with DNA extracted from strainsisolated from CDC ELITE test samples (L. dumoffii, L. longbeachae, L.feelei, and L. cherii) used as experimental samples. To obtainLegionella DNA, colonies were swabbed from bacterial media plates andagitated into water within a biosafety cabinet for extraction using theMoBio Ultraclean DNA extraction kit. DNA was diluted 1/100 in dH₂O priorto analysis and with plastic reagents from Bio-Rad used in the qPCR run.Commercially purchased L. pneumophila genomic standards served as thematerial for the 5-log standard dilutions.

Efficiency of the run was 95.7% with an R̂2 of 0.999, suggesting thatdespite a slightly higher concentration of mix and primers, the run waswell-within acceptable qPCR norms. As seen in FIGS. 1A and 1B, L.pneumophila and L. dumoffii appeared to be preferentially amplifiedrelative to other Legionella bacterial samples. Negative controls P.denitrificans and a non-template added control failed to have meaningfulamplification, with P. aeruginosa amplified at 1×10¹ cfu/ml range. Giventhat the cells used in the extraction were straight from lateexponential/stationary phase cells, this count is likely not an accuratemeasurement of P. aeruginosa cell presence, but rather indicative oflow-level cross contamination. Of particular interest is the highreactivity of L. dumoffii with the qPCR ompS primers.

Repetition of this experiment involves normalizing the DNA inputfollowing enumeration of live cells to get approximately equivalentgenomic units/ml and assist in confirming that this high degree ofsimilarity is accurate.

Example 3: Hybridization Method for Broad Legionella Detection

Following what observed to be a highly selective Legionella primerdesign and a determination that primers (Table 1) should be spacedfurther apart for the proposed hybridization approach, new primers wereselected from a more central to 3′ end of the ompS gene (see FIGS. 2 and7). Specific regions were isolated based on high degree of sequencehomology to L. longbeachae. The “gc” on the primer name refers to“generated complement”, where the primer manufacturer (IDT) generated aprimer that was located on the same location on the opposite strand ofDNA so that the same region of DNA could be tested with primers both 5′and 3′ to its location. The suitability of each primer set was thentested using the conditions listed in Table 2.

To test these primers, genomic DNA from swabbed Legionella strains (orliquid cultures in the case of P. aeruginosa and P. denitrificans) wereassayed with the ompS primers 327, 528, 660, and 825 along with their“gc” counterparts (SEQ ID NOs: 3-10, respectively). The meltingtemperature for each reaction was 45° C. or 40° C. depending on primermelting temperature requirements. For this initial testing reactionconditions were as follows: 1 μM primer, 3 min 95° C., 35 cycles ofdenaturation for 30 s at 94° C.; annealing for 30 s at 40° C. or 45° C.,and elongation for 30 s at 72° C. with a 10 min 72° C. extension step atthe end. The results are displayed in FIGS. 3A through 3F. Given thecleanliness of the reactions, the primer pair 528/825gc SEQ ID NOs: 4and 10 were selected for additional analysis.

Once these reactions had been done, additional work to compare theseprimers to other known sequences, e.g., primers SEQ ID NOs: 15 and 16(ref. 8) were performed to determine whether the primers SEQ ID NO: 4and 10 provided an improvement in Legionella detection. The testing fellwithin two approaches—testing of primers against purified genomic DNAand testing of mock-samples provided by the US CDC for ELITEcertification testing. The results of these tests are displayed in FIGS.4 and 5. Improvement with the inventors' primers was unclear whenassaying the CDC ELITE samples. However, when the same test was runagainst purified DNA, there was an improvement observed incross-reactivity. Primers 528 and 825gc showed less cross-reactivitywith non-Legionella microbial contamination, e.g., P. aeruginosa, thatthe other sequences known in the art (See FIGS. 6A and 6B).

Following this initial screen, the testing protocol was amended. Thetest concentrations of DNA with the defined genomic DNA may have beentoo high to accurately determine if there was an improvement with theinventors' primers relative to the primers SEQ ID NOs. 15 and 16. Basedon interactions with cooling water services companies and field trialexperiences, typical bacterial burden in these samples tended to bearound 1×10⁵ cfu/ml. Second, the primer pool was extended to include twoother sets of known primers from references 10 and 11, SEQ ID NOs: 11through 14 of Table 1. Specific microbial species were tested based onpublications (see e.g., refs. 1-3) and discussions with water treatmentapplications specialists, and availability of these contaminatingmicrobial strains.

A master block of isolated DNA with the equivalent genomic unit/ml tocounted cfu/ml was generated as described in Example 1 and used for PCRanalysis. The degree to which cross-reactivity was observed in eithertotal non-target samples or samples with a high non-target to L.pneumophila DNA ratio was then observed. See FIGS. 8A-8F. The 528F and825gc reverse primers were tested at the beginning and end of theexperiment to ensure that any cross-reactivity observed with otherprimers were genuine and not due to inadvertent cross-contamination ofmaterial. The first round of primer testing resulted in gels too faintto read clearly. FIGS. 8E and 8F show both copies of the experiment.When these tests are repeated, it is anticipated that the results willbe consistent with FIGS. 8E and 8F.

Interestingly, a non-specific amplification was observed with theseprimers against B. cepacia (FIGS. 9A and 9B) and E. aerogenes (FIGS. 9Cand 9D), which have been highlighted with a box. These resultsimmediately suggest that the 528F and 825gc primers constitute animprovement over the known primers. A similar improvement was notobserved with the K. pneumonia tests.

The known 1126R/450 primers SEQ ID NOs. 14 and 13 were assayed accordingto the conditions listed in Table 2. Although data on the performance ofthe 1126R/450 primers against Klebsiella were absent, there was not aclear diminution of performance relative to the 528F/825gc primers withthe species tested with regards to cross-reactivity. The primersappeared to have a higher threshold for detection (1×10²-1×10³) ofLegionella. Some wells had failure to amplify; therefore this experimentwhen repeated is anticipated to confirm a detection sensitivityimprovement.

Finally, the primers SEQ ID NO: 15 and 16 were tested per conditions inTable 2 to determine performance relative to the inventors' primers.

In summary, while the qualities of gels were initially poor and will berepeated, there was a clear cross-reactivity observed against B. cepacia(FIGS. 11A and 11B). Again, this suggests that the DMC primers have animprovement relative to the US20120171661 primers.

However, the present data on these primers provides evidence indicatingthat the inventor-identified primers constitute an improvement of otherompS or mompS-focused primers for the detection of Legionella in acomplex sample.

Technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs and by reference to published texts, which provide oneskilled in the art with a general guide to many of the terms used in thepresent application. Any definitions are provided for clarity only andare not intended to limit the claimed invention.

It should be understood that while various embodiments in thespecification are presented using “comprising” language, under variouscircumstances, a related embodiment is also be described using“consisting of” or “consisting essentially of” language. It is to benoted that the term “a” or “an”, refers to one or more, for example, “atest compound,” is understood to represent one or more test compounds.As such, the terms “a” (or “an”), “one or more,” and “at least one” isused interchangeably herein.

Numerous modifications and variations of the embodiments illustratedabove are included in this specification and are expected to be obviousto one skilled in the art. Such modifications and alterations to thecompositions and processes described herein are believed to beencompassed in the scope of the claims appended hereto. All documents,including patents, patent applications and publications, and non-patentpublications listed or referred to above, as well as the attachedfigures and/or Sequence Listing, are incorporated herein by reference intheir entireties to the extent they are not inconsistent with theexplicit teachings of this specification.

What is claimed is:
 1. A reagent for examining a water supply formicrobial contamination comprising with nucleotide sequence having highspecificity for a Legionella microbial species, wherein said nucleotidesequence does not cross-react, or minimally cross-reacts, with amicrobial species other than Legionella.
 2. The reagent according toclaim 1 comprising at least one nucleotide sequence primer comprisingSEQ ID NO: 1 through 10 or a combination of multiple primer sequencescomprising SEQ ID NO:1 through 10 or modifications of SEQ ID NO: 1through
 10. 3. The reagent according to claim 2 comprising a set ofprimers comprising a forward nucleotide sequence primer and a reversenucleotide sequence primer.
 4. The reagent according to claim 3, whereinthe forward primer comprises at least one of SEQ ID NO: 1, 3, 4, 5 or 6and the reverse primer comprises at least one of SEQ ID NO: 2, 7, 8, 9,or
 10. 5. The reagent according to claim 3, wherein the forward primercomprises SEQ ID NO: 4 and the reverse primer comprises SEQ ID NO: 10.6. The reagent according to claim 2 comprising multiple sets of reagentsselected from the primers comprising SEQ ID NOs: 1-10.
 7. The reagentaccording to claim 2, wherein the reagent comprises all nucleotidesequence primers comprising SEQ ID NO: 1-10.
 8. The reagent according toclaim 2, which further comprises a substrate upon which one or more ofthe nucleotide sequences are immobilized or fixed.
 9. The reagentaccording to claim 2, wherein a detectable label or label component isassociated with at least one primer.
 10. The reagent according to claim8, wherein said substrate is an array, a microarray, a microchip, aplastic surface, or a glass surface.
 11. The reagent according to claim1, wherein the nucleotide sequence is RNA.
 12. The reagent according toclaim 1, wherein the nucleotide sequence is DNA.
 13. A kit comprisingthe reagent of claim 1 and one or more components selected from a label,a substrate, a label component capable of interacting with the label andgenerating a detectable signal.
 14. A water monitoring device comprisinga reagent of claim
 1. 15. A method of examining a water supply formicrobial contamination comprising: contacting a water supply with areagent of claim 1 having high specificity for a Legionella microbialspecies, wherein said reagent does not cross-react, or minimallycross-reacts, with a microbial species other than Legionella; anddetecting, or measuring, a contaminating concentration of a Legionellaspecies in the water supply.
 16. The method according to claim 15,wherein the reagent comprises at least one nucleotide sequence primerselected from SEQ ID NO: 1 through 10, a combination of primer sequencescomprising SEQ ID NO: 1 through 10, or modifications of SEQ ID NO: 1through
 10. 17. The method according to claim 15, wherein measuringcomprises annealing a primer or a combination of primers to a targetednucleic acid sequence present in Legionella at a selected annealingtemperature lower than 58° C.
 18. The method according to claim 15,wherein the measuring comprises hybridizing a primer to a targetednucleic acid sequence present in Legionella.
 19. The method according toclaim 15, wherein the measuring comprises performing a polymerase chainreaction (PCR) or real time qualitative polymerase chain reaction(qPCR).
 20. The method according to claim 15, comprising distinguishingbetween contamination with Legionella species from contamination with aPseudomonas species.